WTAP-mediated m6A modification of lncRNA Snhg1 improves myocardial ischemia-reperfusion injury via miR-361-5p/OPA1-dependent mitochondrial fusion

Background Myocardial ischemia-reperfusion injury (MIRI) is caused by reperfusion after ischemic heart disease. LncRNA Snhg1 regulates the progression of various diseases. N6-methyladenosine (m6A) is the frequent RNA modification and plays a critical role in MIRI. However, it is unclear whether lncRNA Snhg1 regulates MIRI progression and whether the lncRNA Snhg1 was modified by m6A methylation. Methods Mouse cardiomyocytes HL-1 cells were utilized to construct the hypoxia/reoxygenation (H/R) injury model. HL-1 cell viability was evaluated utilizing CCK-8 method. Cell apoptosis, mitochondrial reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) were quantitated utilizing flow cytometry. RNA immunoprecipitation and dual-luciferase reporter assays were applied to measure the m6A methylation and the interactions between lncRNA Snhg1 and targeted miRNA or target miRNAs and its target gene. The I/R mouse model was constructed with adenovirus expressing lncRNA Snhg1. HE and TUNEL staining were used to evaluate myocardial tissue damage and apoptosis. Results LncRNA Snhg1 was down-regulated after H/R injury, and overexpressed lncRNA Snhg1 suppressed H/R-stimulated cell apoptosis, mitochondrial ROS level and polarization. Besides, lncRNA Snhg1 could target miR-361-5p, and miR-361-5p targeted OPA1. Overexpressed lncRNA Snhg1 suppressed H/R-stimulated cell apoptosis, mitochondrial ROS level and polarization though the miR-361-5p/OPA1 axis. Furthermore, WTAP induced lncRNA Snhg1 m6A modification in H/R-stimulated HL-1 cells. Moreover, enforced lncRNA Snhg1 repressed I/R-stimulated myocardial tissue damage and apoptosis and regulated the miR-361-5p and OPA1 levels. Conclusion WTAP-mediated m6A modification of lncRNA Snhg1 regulated MIRI progression through modulating myocardial apoptosis, mitochondrial ROS production, and mitochondrial polarization via miR-361-5p/OPA1 axis, providing the evidence for lncRNA as the prospective target for alleviating MIRI progression.


Background
Ischemic heart disease (IHD) is a main cause of death around the world, with high mortality and morbidity [1,2].Myocardial infarction is the most popular primary symptom of IHD [3].In clinical therapy, prompt reperfusion is crucial to decrease infarct size, save the ischemic myocardium, and prevent the onset of heart failure [1,4].Nevertheless, reperfusion may induce myocardial injury named myocardial ischemia/reperfusion injury (MIRI) [4][5][6].Increasing evidence reveals that reperfusion injury accounts for 50% of the approximate myocardial infarct size [7,8].MIRI leads to significantly increased mortality in myocardial infarction [9].Thus, MIRI is a primary risk factor threatening global health.It is significant to clarify the mechanism of the MIRI progression and explore the potential therapy strategy to limit myocardial injury.
Mitochondrial dysfunction is closely related to metabolic disorders, ischemic heart disease, and many other diseases [10].During MIRI, mitochondria ATP production and mitochondrial membrane potential (MMP) are decreased, while reactive oxygen species (ROS) are excessively produced, which collectively lead to myocardial dysfunction, DNA damage, and apoptosis [11].In the process of apoptosis, mitochondrial fusion is observed, which is regulated by several proteins including optic atrophy protein 1 (OPA1) and mitofusin 1 (MFN1) and 2 (MFN2) [12].Therefore, it is important to identify potential factorsthat modulate mitochondrial dynamics to prevent MIRI.
Long non-coding RNAs (lncRNAs) belong to the noncoding RNAs (ncRNA) family [13].The lncRNAs are over 200 nt in length without protein-coding capacity but usually modulate gene expressions at the post-transcriptional level via competing endogenous RNAs (ceR-NAs) mechanism [14].The ceRNA mechanism proposes that lncRNAs may serve as ceRNA sponges to absorb microRNAs (miRNAs), thereby mitigating the inhibitory action of miRNAs on target mRNAs [15].As a member of the lncRNAs family, lncRNA SNHG1 has been extensively studied in a variety of diseases, including MIRI.For example, lncRNA SNHG1 in HL-1 cells was significantly reduced by H/R treatment and assuaged HL-1 cell pyroptosis by regulating the KLF4/TRPV1/AKT axis through sponging miR-137-3p [16].lncRNA SNHG1 expression was downregulated in H/R-induced H9c2 cells and SNHG1/miR-16-5p/GATA4 regulated H9c2 cell apoptosis induced by H/R [17].lncRNA SNHG1 expression in H/Rinduced AC16 cells was significantly decreased and SNHG1/miR-450b-5p/IGF1 axis inhibited the apoptosis and oxidative stress levels of H/Rinduced AC16 cells [18].Moroever, knockdown of SNHG1 alleviated mitochondrial dysfunction in cerebral ischemia/reperfusion injury [19].However, whether lncRNA SNHG1 may regulate cardiomyocyte apoptosis and mitochondrial dysfunction in MIRI progression by sponging miRNA is still poorly understood.
N6-methyladenosine (m 6 A) is the frequent RNA modification in eukaryotic cells at the posttranscriptional level [20,21].The potential regulatory efect of m6A modifcation may infuence the occurrence and development of a variety of cardiovascular diseases [22].Studies have shown that Wilms tumor 1-associated protein (WTAP) is a RNA methyltransferase that promotes myocardial I/R injury progression and mediates lncRNA m6A modifcation through m 6 A reader [23,24].Prediction analysis by SRAMP database revealed multiple m6A modifcation sites in lncRNA SNHG1 sequence.However, whether lncRNA SNHG1 was modified by WTAP-mediated m 6 A methylation in MIRI is still poorly understood.
This study was therefore conducted to investigate the role of WTAP-mediated m 6 A methylation of lncRNA Snhg1 in myocardial injury triggered by mitochondrial dysfunction after MIRI.We showed for the first time that lncRNA Snhg1 sponged miR-361-5p to upregulate the expression of OPA1, which then activated the mitochondrial fusion to attenuate MIRI.Moreover, WTAP induced lncRNA Snhg1 m6A modification and inhibited lncRNA Snhg1 stability by m 6 A reader YTH N6-methyladenosine RNA-binding protein 2 (YTHDF2).The results of the present study may provide a theoretical basis for future clinical studies and highlight potential targets for the treatment of MIRI.

Cell viability assay
Forty-eight hours after treatment, cells were dispensed into 96-well plates with 3 × 10 3 cells each well.Twentyfour hours later, cells were added to 10 µL of CCK-8 for 1 h incubation.The absorbance was recorded at 450 nm utilizing the microplate reader.

Measurement of ATP
ATP level was evaluated using an ATP assay kit (Jiancheng, Nanjing, China) following the supplier's instructions.HL-1 cells were plated into the 6-well plate for24 h.After that, HL-1 cells were collected utilizing lysis buffer.The detection solution was then introduced to the supernatant and ATP level was tested via normalizing to the total protein content.

Quantitative real-time PCR (RT-qPCR)
RNAs samples of HL-

Analysis of m 6 A content
RNAs samples were acquired utilizing Trizol (Invitrogen).The purification of Poly(A) + RNA was performed utilizing GenElute™ mRNA Miniprep Kit (Sigma, Louis, MO, USA).The m 6 A level was estimated utilizing m 6 A RNA Methylation Assay Kit (Abcam; ab185912) as previously described [28].

Dual-luciferase reporter assay
Putative target regions of miR-361-5p to lncRNA Snhg1 and Opa1 3'-UTR were predicted using Starbase and TargetScan.Predicted sequences of both wild type and mutant were synthesized, respectively, and then inserted into luciferase reporter vectors (PmirGLO), the vectors were then nominated as WT 3'-UTR and Mut 3'-UTR.The pGL3-lncRNA Snhg1 or pGL3-Opa1 3'-UTR and pRL-TK renilla (Promega) luciferase reporter vector were introduced into HL-1 cells accompanied by miR-361-5p mimic or inhibitor.The sequence of lncRNA Snhg1 containing the m 6 A motifs was generated by Generay Technologies (Shanghai, China) and cloned into the upstream of the pGL3-basic firefly luciferase vector.After that, the pGL3-lncRNA Snhg1 and pRL-TK renilla were co-transfected into the HL-1, which were stimulated by H/R and introduced into Wtap silencing or Wtap-expressing plasmid.The luciferase activities were recorded utilizing the dual luciferase reporter gene kit (Yuanpinghao, Beijing, China) 48 h after transfection.

Determination of mRNA stability
HL-1 cells were incubated with 0.2 mM actinomycin D (Selleck, Shanghai, China).At the time point of 0, 3, and 6 h, total RNAs were isolated and produced cDNA utilizing the oligo(dT) primer.The mRNA expressions were quantified utilizing RT-qPCR.

Animal experiment and drug administration
C57/BL6 mice aged 6-7 weeks (male, 20-25 g) were obtained from the SLAC Laboratory Animal Center of Shanghai (Shanghai, China).The mouse myocardial I/R model was constructed following previously described [29].Recombinant adenovirus expressing lncRNA Snhg1 (2 × 10 10 pfu/ml) or blank pShuttle-CMV vector was introduced into the left ventricular anterior wall via injection 24 h prior to I/R (four injections of 30 µl each with an interval of 4′30″ between injections).At 24 h after I/R, the cardiac function was evaluated using the Vevo 2100 Imaging (Visual Sonics, Toronto, Canada) echocardiographic system equipped with a 30 MHz transducer and left ventricular ejection fraction and left ventricular fractional shortening were calculated [30].Heart rate, left ventricle end-diastolic pressure and left ventricle endsystolic pressure were recorded using an AcqKnowledge version 3.8.

Histological analysis and TUNEL staining
The myocardium was excised to conduct hematoxylin and eosin (HE) and terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) staining as previously described [32].Heart tissues were fixed, embedded, and cut into 5-µm sections.The sections were placed on glass slides, deparaffinized, and stained sequentially with hematoxylin and eosin (RichardAllan Scientific Co., Kalamazoo, MI, USA).The stained tissue sections were analyzed using a light microscope (Axio Imager M1; Carl Zeiss AG).Paraffin-embedded heart sections were used for apoptosis determination using TUNEL staining.Briefly, the slides were first incubated in 50 µg/mL proteinase K solution at 37 °C for 30 min.Then, followed by three times washing in PBS, slides were incubated with the TUNEL detection buffer at 37 °C for 1 h.Finally, the images were obtained and analyzed using the Olympus Fluoview FV300 version.

Measurement of CK-MB and cTnT levels
The serum levels of mouse creatine kinase myocardialband (CK-MB; X-Y Biotechnology; XY9M0929) and myocardial troponin-T (cTnT; X-Y Biotechnology; XY9M0582) were estimated using ELISA kits and analyzed to assess the degree of myocardial damage.

Data analysis
All experiments and assays were conducted with three replicates, and quantitative data were described as the mean ± standard deviation.Data analysis was achieved utilizing GraphPad Prism 8.4.2.Unpaired t-test and one-way ANOVA followed by Dunnett's multiple comparisons test were selected to confirm group differences.P < 0.05 was designated as statistically significant.

Overexpressed LncRNA Snhg1 restrained H/R-stimulated cell apoptosis, mitochondrial ROS production, and mitochondrial polarization
To clarify the action of lncRNA Snhg1 on MIRI, HL-1 cells were selected to construct the H/R model.The lncRNA Snhg1 level in the H/R model was first measured.It was found that lncRNA Snhg1 was down-regulated after H/R injury and gradually decreased with prolonged re-oxygenation time (Fig. 1A).Therefore, the effects of lncRNA Snhg1 overexpression on H/R-induced injury were next investigated after lncRNA Snhg1 overexpressing lentiviral transduced into HL-1 cells.Results showed that H/R injury decreased the HL-1 cell viability, which was partially restored by enforced lncRNA Snhg1 (Fig. 1B).Besides, H/R injury accelerated HL-1 cell apoptosis but was restrained by overexpressed lncRNA Snhg1 (Fig. 1C and 1D).In addition, mitochondrial ROS level was increased in H/R-stimulated HL-1 cells, while overexpressed lncRNA Snhg1 suppressed this phenomenon (Fig. 1E and 1G).Furthermore, H/R injury decreased the MMP and ATP level of HL-1 cells, which was increased after overexpression of lncRNA Snhg1 (Fig. 1F, 1H and 1I).Moreover, the down-regulated lncRNA Snhg1 caused by H/R challenge was restored by lncRNA Snhg1 overexpressing lentiviral (Fig. 1J).Therefore, overexpressed lncRNA Snhg1 suppressed H/R-stimulated cell apoptosis, mitochondrial ROS production, and mitochondrial polarization in HL-1 cells.

LncRNA Snhg1 silencing promotes H/R-stimulated cell apoptosis, mitochondrial ROS production, and mitochondrial polarization via targeting miR-361-5p
To elucidate the precise mechanism of lncRNA Snhg1 on MIRI progression, shSnhg1 lentivirus, and miR-361-5p inhibitor were introduced into H/R-induced HL-1 cells.Results showed that H/R treatment suppressed cell viability of HL-1 cells, and silencing of lncRNA Snhg1 exacerbated H/R-induced injury on cell viability, while this phenomenon was reversed after miR-361-5p inhibitor treatment (Fig. 3A).Besides, the promotion effect of silenced lncRNA Snhg1 on H/R-stimulated cell apoptosis was restrained by miR-361-5p inhibitor (Fig. 3B and 3D).

WTAP induces lncRNA Snhg1 m 6 A modification
To illustrate whether lncRNA Snhg1 was modified by m 6 A methylation, the shWtap lentivirus and Wtap overexpression lentivirus were introduced into H/Rinduced HL-1 cells, which restrained and enhanced the Wtap level, respectively (Fig. 6A and 6C).MeRIP-PCR assay proved the elevated m 6 A level of lncRNA Snhg1 in H/R-stimulated HL-1 cells, while silenced Wtap drastically decreased the m 6 A level of lncRNA Snhg1 (Fig. 6D).Conversely, overexpressed Wtap increased the m 6 A level of lncRNA Snhg1 (Fig. 6D).Luciferase assay showed that lncRNA Snhg1 m 6 A activity was enhanced in H/R-induced HL-1 cells, while silenced Wtap drastically decreased the lncRNA Snhg1 m 6 A activity (Fig. 6E).
Overexpressed WTAP increased the lncRNA Snhg1 m 6 A activity (Fig. 6E).After that, whether m 6 A modification on lncRNA Snhg1 affected its expression was determined.It was observed that silenced Wtap enhanced the lncRNA Snhg1 expression, but enforced Wtap suppressed the lncRNA Snhg1 expression (Fig. 6F).Besides, the RNA stability of lncRNA Snhg1 was measured after silenced Wtap and treated with actinomycin D. Results showed that silenced Wtap increased the stability of lncRNA Snhg1 (Fig. 6G).Additionally, the effect of m 6 A "reader" YTHDF2 on lncRNA Snhg1 expression was studied after transfection of shYthdf2.The shYthdf2 considerably silenced the Ythdf2 expression (Fig. 6H  and 6J).The lncRNA Snhg1 was elevated after silencing Ythdf2 (Fig. 6K).Silenced Ythdf2 increased the stability of lncRNA Snhg1 (Fig. 6L).Furthermore, the RIP-PCR assay found that lncRNA Snhg1 was obviously enriched using the anti-YTHDF2 antibody (Fig. 6M).Overall, these results suggested that WTAP induced lncRNA Snhg1 m 6 A modification.

LncRNA Snhg1 overexpression inhibits I/R-induced myocardial tissue damage and apoptosis
To interpret the action of lncRNA Snhg1 on MIRI progression in vivo, the myocardial I/R mouse model was established with overexpressed lncRNA Snhg1.Echocardiography was performed to measure cardiac function parameters (Fig. 7A).Cardiac functions results showed that I/R treatment significantly reduced cardiac ejection fraction, fractional shortening, left ventricular end systolic pressure and heart rate, and promoted left ventricular end diastolic pressure (P < 0.001), these phenomena were reversed by overexpressed lncRNA Snhg1 (P < 0.001, Table 2).HE staining results found that the myocardial tissue exhibited apparent damage, including disordered structure, inflammatory infiltration, and interstitial edema after I/R treatment, which was alleviated by overexpressed lncRNA Snhg1 (Fig. 7B).The serum levels of CK-MB and cTnT were also increased in I/R mice, which were alleviated by overexpressed lncRNA Snhg1 (Fig. 7C and 7D).Besides, I/R treatment induced myocardial cell apoptosis, but overexpressed lncRNA Snhg1 suppressed this effect (Fig. 7E and 7F).In addition, the lncRNA Snhg1 expression was decreased in myocardial tissue after I/R injury, which was elevated after transfection of lncRNA Snhg1 overexpression lentivirus (Fig. 7G).Furthermore, miR-361-5p was enhanced in the myocardial tissue of I/R mice, and overexpressed lncRNA Snhg1 restrained the miR-361-5p level (Fig. 7H).Moreover, OPA1 were repressed in myocardial tissue after I/R treatment, which was restored by overexpressed lncRNA Snhg1 (Fig. 7I and 7K).To sum up, enforced lncRNA Snhg1 restrained I/R-stimulated myocardial tissue damage and apoptosis and regulated the miR-361-5p and OPA1 levels.

Discussion
Myocardial ischemia-reperfusion injury (MIRI) is an injury caused by the recovery of coronary artery blood flow after ischemic heart disease and increases myocardial infarction [3][4][5][6].LncRNA Snhg1 regulates disease progression by modulating the cell cycle, apoptosis, autophagy, oxygen species, and mitochondrial function [33][34][35].N6-methyladenosine (m 6 A) is the frequent RNA modification manner, and lncRNAs can be modified by m 6 A methylation [22,24].In this research, we elucidated the action and mechanism of lncRNA Snhg1 on MIRI progression and clarified whether the lncRNA Snhg1 was modified by m 6 A methylation.The findings revealed that WTAP-mediated m 6 A modified lncRNA Snhg1 modulated MIRI progression manifested in regulating cell apoptosis, mitochondrial ROS production, and MMP via miR-361-5p/OPA1 axis (Fig. 7L).These results demonstrated the existence of the WTAP/SNHG1/miR-361-5p/ OPA1 regulatory axis and provided a novel therapeutic target for the treatment of ischemic cardiomyocyte injury.LncRNA Snhg1 belongs to the lncRNAs family, and it usually presents abnormal expression in various disease tissues and cells [36][37][38].To elaborate the potential action of lncRNA Snhg1 on MIRI, the lncRNA Snhg1 level was explored in H/R-stimulated HL-1 cells and found that lncRNA Snhg1 was decreased after H/R challenge, which was in line with the reported study [18].The abnormal level of lncRNA Snhg1 implied possible involvement in MIRI progression.As expected, overexpression of lncRNA Snhg1 restored HL-1 cell viability inhibited by H/R challenge.Actually, the regulatory action of lncRNA Snhg1 on cell viability has also been demonstrated in multiple disorders, such as cancers, Parkinson's disease, and hypoxic injury [38][39][40].Besides, apoptosis is a significant feature of MIRI and is considered an essential pathogenic factor in MIRI [3,41].Apoptosis causes cardiomyocyte loss and myocardium remolding and aggravates inflammatory response [3].Therefore, inhibiting myocardial cell apoptosis is critical for limiting MIRI progression [41].This research demonstrated that enforced lncRNA Snhg1 restrained myocardial cell apoptosis in H/R-stimulated HL-1 cells and I/R mice model.Similarly, overexpressed lncRNA Snhg1 was also proved to repress cell apoptosis behavior in H/R-stimulated AC16 cells [18].Lv et al. revealed that enforced lncRNA Snhg1 inhibited oxygen-glucose deprivation-stimulated cell apoptosis [42].Furthermore, it is accepted that mitochondria are key targets and the source of tissue damage, and the structural and functional changes of mitochondria are critical in the pathogenesis of MIRI [10].MIRI could cause early mitochondrial-driven injury with excessive ROS production and calcium regulation disorder, causing abnormal permeation, loss of MMP, and swelling and damage to mitochondria of mitochondrial permeability transition pore [43,44].In the current study, increased mitochondrial ROS production and decreased MMP and ATP levels were exhibited after H/R challenge, which was in line with the reported research [10].Interestingly, these changes in the structural and functional changes of mitochondria caused by H/R challenge were reversed by overexpressed lncRNA Snhg1.In other words, enforced lncRNA Snhg1 suppressed mitochondrial ROS production and mitochondrial polarization in H/R-induced HL-1 cells.To sum up, these findings indicated the effectiveness of lncRNA Snhg1 in improving cell apoptosis, mitochondrial ROS production, and mitochondrial polarization in MIRI progression.
LncRNAs generally regulate disease progression via the ceRNA mechanism [14].To elucidate the precise mechanism of lncRNA Snhg1 on the regulation of cell apoptosis, mitochondrial ROS production, and mitochondrial polarization in MIRI progression, the potential target miRNA of lncRNA Snhg1 was searched, and the findings discovered that lncRNA Snhg1 could target miR-361-5p.So far, this study first reports that lncRNA Snhg1 targeted miR-361-5p.The actions of miR-361-5p on modulating cardiomyocyte apoptosis and mitochondrial function were proved in previous studies [45,46].Li et  [46].The miRNAs usually exerted functions via inhibiting the target gene expression through binding to 3'-UTR [47].Therefore, the target gene of miR-361-5p was explored, and the findings demonstrated that miR-361-5p could target OPA1.OPA1, a GTPase at the mitochondrial inner membrane, exerts a critical function on mitochondrial fusion [12].As expected, the miR-361-5p inhibitor inhibited H/R-stimulated cell apoptosis, mitochondrial ROS level and polarization in HL-1 cells by targeting OPA1.Consistent with our findings, the previous study reported that OPA1 was reduced in cardiomyocytes with simulated I/R challenge, and depletion of OPA1 sensitized the cells to mitochondrial fragmentation, apoptosis, and I/R injury [48].Wang et al. also found that OPA1 attenuated ROS production and mitochondrial dysfunction in H 2 O 2 -challenged H9C2 cells [49].Besides, we also found overexpressed lncRNA Snhg1 restrained the miR-361-5p expression and elevated OPA1 level in the I/R mice model in vivo.Overall, this evidence suggested that lncRNA Snhg1 regulated MIRI progression via the miR-361-5p/OPA1 axis.The m 6 A methylation is the frequent RNA modification manner in eukaryotic cells [20,21].Recent studies discovered that lncRNAs could be modified by m 6 A methylation [22,24].Jiang et al. reported that METTL3 induced the lncRNA Snhg1 m 6 A modification and improved the stability of the lncRNA Snhg1 in non-small cell lung cancer [50].Therefore, whether lncRNA Snhg1 was modified by m 6 A methylation in HL-1 cells after H/R challenge was studied in this study and it was proved that WTAP induced lncRNA Snhg1 m 6 A modification and YTHDF2 was a specific m 6 A reader of lncRNA Snhg1 in HL-1 cells with H/R challenge.These findings suggested that WTAP-mediated m 6 A is associated with the expression of lncRNA Snhg1, probably through regulating lncRNA Snhg1 stability, which provided new answers to the question of why lncRNA Snhg1 exhibited abnormal expression after H/R injury.

Conclusions
WTAP-mediated m 6 A modification of lncRNA Snhg1 regulated MIRI progression through modulating myocardial apoptosis, mitochondrial ROS production, and mitochondrial polarization via the miR-361-5p/Opa1 axis, providing the evidence for lncRNA as the prospective target for alleviating MIRI progression.

Table 2
Cardiac functions for experiment animals